Programmed cell death has been shown to be an essential feature of negative selection of autoreactive lymphocytes and regulation of both physiological and pathological immune responses. Fas, also known as CD95, is a member of the TNF-receptor superfamily and has been shown to be important in apoptosis of activated T and B lymphocytes initiated by signaling through their antigen receptors. Humans and mice with germ line dominant-negative mutations in Fas accumulate abnormal lymphocytes and develop systemic autoimmunity similar to patients with Systemic Lupus Erythematosus (SLE). While most patients with non-familial autoimmune disease do not carry Fas mutations, there is evidence that Fas-mediated apoptosis may be impaired in the milieu of chronic inflammation. We are investigating which signals regulate Fas-mediated apoptosis in T cells, with the eventual aim of harnessing these discoveries to modulate Fas-induced apoptosis for therapeutic goals in human disease. In activated CD4+ T cells, TCR restimulation triggers apoptosis that depends in large part on interactions between the death receptor Fas and its ligand FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but it is not known which pathways mediate this process. Using a variety of approaches, we are investigating molecular and cellular mechanisms regulating the TCR- and Fas-induced apoptosis pathways. We have found considerable heterogeneity in the ability of various T cell subsets to undergo Fas-mediated apoptosis and are investigating the molecular mechanisms underlying this heterogeneity. The goal in understanding these mechanisms is to design specific therapies to sensitize autoreactive lymphocytes to Fas-mediated apoptosis, which could constitute a long-acting and potentially permanent treatment for various autoimmune diseases such as SLE, Multiple Sclerosis, Rheumatoid Arthritis, Type-I diabetes, and others in which autoreactive lymphocytes play a role. Through collaborations with investigators at NIH studying patients with the Autoimmune Lymphoproliferative Syndrome (ALPS), a rare disorder associated with dominant-interfering Fas mutations, and the more common polygenic autoimmune disease SLE, we are investigating translational implications of these findings. We are also investigating the subcellular trafficking of Fas Ligand (FasL), the TNF-family cytokine ligand for Fas. In addition to trafficking to the plasma membrane as a type II transmembrane protein, FasL is known to be sorted into secretory lysosomes, where it can be secreted in vesicles and cleaved by metalloproteases. We are investigating which forms of FasL participate in RICD, and which molecules and motifs within the FasL cytoplasmic N-terminal domain direct its trafficking to secretory lysosomes. Our recent discovery that lipid raft association of Fas is crucial for apoptotic signaling has re-focused attention on the oligomerization state of the receptor before and after ligand binding, and recent breakthroughs in superresolution microscopy have made imaging the signaling complexes of Fas and related receptors at single molecule resolution possible. We used the PALM super-resolution technique in collaboration with Prabuddha Sengupta to study the effects of specific Fas subdomains on receptor oligomerization before and after ligand engagement. The Fas C199V mutation did not change the oligomerization state of Fas prior to ligand engagement, but instead, prevented formation of higher order oligomers of receptors upon incubation with an agonistic antibody. Mutagenesis studies of FasL in mice have shown that the transmembrane. We developed a system where trimerized FasL is anchored in a lipid bilayer, allowing us to revisit the requirements of different domains of Fas for receptor clustering. Deletion of the N-terminal pre-ligand assembly domain (PLAD), which reduces ligand binding as well as receptor pre-association, produced a partial defect in the formation of larger receptor oligomers. To confirm these findings, we also used Fluorescence Resonance Energy Transfer (FRET). We have continued studies of Fas apoptotic and non-apoptotic signaling on tumor immunotherapy by CD8+ T cells. We transduced T cells with Fas constructs containing dominant negative mutations derived from ALPS patients, or lacking the death domain all together. In studies with a TCR directed against melanoma antigens and T cells transduced with a Chimeric Antigen Receptor (CAR) against CD19 in the setting of CD19(+) mouse lymphoma model, we found that the dominant-negative Fas protected against TCR and FasL induced cell death in vitro and in vivo, and significantly enhanced the efficacy of tumor immunotherapy.